PROJECT SUMMARY/ABSTRACT Mitochondria are centers of metabolism whose activities need to be calibrated to meet changing cellular needs. General dysfunction of these organelles is implicated in many common human disorders, including Parkinson?s, Alzheimer?s, various cancers, metabolic syndrome, type 2 diabetes, obesity, non-alcoholic fatty liver disease, heart failure, and general metabolic inflexibility, most often through unclear means. Defining the pathogenic mitochondrial alterations that contribute to these metabolic disorders and devising new therapeutic strategies to rectify them represent principal challenges in mitochondrial medicine. A potential contributor to this dysfunction is aberrant intra-mitochondrial protein phosphorylation?a process recognized as critical for pyruvate dehydrogenase regulation for more than 50 years, but relatively unexplored otherwise. Recent efforts from our laboratories and others have now revealed that mitochondrial proteins are replete with dynamic phosphorylation that changes reproducibly between healthy and diseased states, and that phosphorylation can alter the activities of proteins involved in core metabolic pathways. We have also now connected select phosphorylation events to poorly characterized matrix protein phosphatases, thereby beginning to establish a mechanistic framework for understanding mitochondrial protein phosphorylation and its effects on metabolic activities. Given these emerging findings, the premise of this project is that reversible phosphorylation may be widely important in calibrating mitochondrial metabolism, and that its mismanagement could contribute to the pathophysiology of mitochondria-related disorders. Rigorous new efforts to reveal how phosphorylation affects mitochondrial protein function and to define the phosphatases that target each site may ultimately enable a new therapeutic strategy focused on manipulation of the mitochondrial phosphorylation network. The work proposed here is designed to take significant steps toward these goals. In particular, the contributions of our efforts will be 1) to define the physiological functions and direct biochemical substrates of Pptc7, a poorly characterized mitochondrial matrix phosphatase whose disruption causes a dysregulated fatty acid oxidation (FAO) and neonatal death, 2) to establish the mechanistic effects of phosphorylation on putative Pptc7 substrates of outstanding importance to FAO and protein import, and 3) to begin systematically connecting an extended set of orphan mitochondrial phosphatases to candidate substrates and metabolic processes, thereby opening up a largely untapped area of mitochondrial metabolic regulation. Altogether, through a comprehensive approach that combines mammalian physiology, omics-level analyses, and rigorous biochemistry, we aim to make definitive connections between mitochondrial phosphatases and their substrates, establish a broad framework for understanding the role of this post-translation modification in calibrating mitochondrial activities, and ultimately pave the way for a new therapeutic strategy to rectify mitochondrial dysfunction.